I am trying to express a kinase domain of a protein. The construct is in pET-His10 TEV LIC vector and I am using pLysS cells. The cells were induced with 4mM IPTG and expressed at 22C. When I started to harvest (Pelleting down) the cells (8000 rpm for 10 min) I am getting slimy pellet (not like the usual pellet that stick to the bottom of the centrifuge tube). I increased the time for pellet by 20 min but still I am getting the slimy pellet.
What could be reason for the same?
Most likely cell lysis. Try expressing in a "normal" (i.e. non pLysS) strain.
hi
There are three possibilities :
1- cell lysis by high speed Centrifugation >> you can used 4000 RPM 10min 4c
2- cell lysis by bacteriophage infection >> transform new and fresh E.coli Bach
3- cell cytotoxicity by recombinant protein expression >>
A-tray to check other host
B- decrease IPTG concentration ( for example 1mM ) or reduce incubation temperature ( for example 17 c)
good luck
Dear Subhashish
did you measure the od(600nm) at the end of the culture?
at which OD(600) due you performed induction with IPTG? Why a high IPTG concentration?
is it possible that have cell lysis due to toxycity of your protein or phage contamination but i suspect that you have small pellet just because, low temperature, high IPTG and second antibiotic to mantain the plyss , all togheter just result in poor bacterial growth and you need to review your growth/induction protocoll.
ciao
Manuele
Thank you all.
Manuele Martinelli I had induced at a range of 0.9-1OD.
To start with just scan various iptg concentrations and induction time ( 4mM iptg is high, do you really need od 1 ? induce with iptg when cells are in log phase )
Hi Shubhashish,
I think your cell may not grow well, probably because of phage contamination or the toxicity of the expressed target protein.
In addition, I don’t think the induction conditions you used are appropriate and I have the same question as others, that is, I don’ understand why you use such a high concentration of IPTG. Usually, I use IPTG with a final concentration of 0.5mM.
All the best!
Sorry, their was a typing error. I use 0.4mM of IPTG and not 4mM
Reza Hadavi , Can you please elaborate on bacteriophage infection? How do we determine if there is any bacteriophage infection in our cells? Will there be any change in the cell pellet formation?
Also, if someone can please help me understand that why cell pellet in 10% glycerol appears flaky sometimes? Should I be worried or has any of you encountered it before?
Thanks again
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